Transgenic mice harboring the Simian Virus 40 gene encoding the large tumor antigen (T antigen) develop tumors of tissues specified by the enhancer-promoter regulating this gene. (1) Two domains of the T antigen have been identified as essential to immortalization and transformation of cells in culture; (a) amino acid residues 1-120, which is known to bind to the retinoblastoma protein (RB), and (b) amino acid residues 325-625, which is known to bind to the p53 protein. Genetic constructs (fusion proteins or mutant T antigens) have been designed that encode stable protein domains for T antigen 1-120 and T antigen 325-625. The phenotypes of each of these expression vectors will be tested separately and together in cell culture (cotransfection) or in vivo (Fl mice). By employing a diverse set of enhancer-promoters in vivo (insulin, elastase, albumin), the effects of binding the RB protein and/or p53 to the T antigen domain can be examined. (2) The degree to which a small number of precursor cells contribute to a T antigen induced tumor (clonality) in transgenic mice will be tested by creating allophenic or tetraparental mice from two different transgenic mice harboring the same transgenic (T antigen) at different integration sites. Southern blots that measure the contribution of the different integrants would provide a qualitative measure of the contribution of each cell type to a segment of the tumor. (3) A genetic analysis of different inbred strains of mice harboring the same transgene has indicated that NZW mice can negatively regulate, in a dominant fashion, the expression of the SV40 T antigen transgene. Studies which map, characterize and identify this negative regulatory element are described. (4) All studies to-date employing transgenic mice expressing the SV40 large T antigen have indicated that a second event, in addition to T antigen expression, is required for tumorigenesis. Experiments are designed herein to measure whether the expression of T antigen increases the mutation frequency in transgenic mice. (5) The SV40 large T antigen structural gene itself contains some nucleotide signals that focuses expression of this gene to the choroid plexus. Experiments have been designed to identify and map this signal and isolate the proteins of the choroid plexus that recognize it.
